, Volume 68, Issue 4, pp 687–700 | Cite as

Evaluation of cytotoxic activities of snake venoms toward breast (MCF-7) and skin cancer (A-375) cell lines

  • Michael J. Bradshaw
  • Anthony J. Saviola
  • Elizabeth Fesler
  • Stephen P. MackessyEmail author
Original Research


Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical activities. This wide array of pharmacologies associated with snake venoms has made them attractive sources for research into potentially novel therapeutics, and several venom-derived drugs are now in use. In the current study we performed a broad screen of a variety of venoms (61 taxa) from the major venomous snake families (Viperidae, Elapidae and “Colubridae”) in order to examine cytotoxic effects toward MCF-7 breast cancer cells and A-375 melanoma cells. MTT cell viability assays of cancer cells incubated with crude venoms revealed that most venoms showed significant cytotoxicity. We further investigated venom from the Red-bellied Blacksnake (Pseudechis porphyriacus); venom was fractionated by ion exchange fast protein liquid chromatography and several cytotoxic components were isolated. SDS-PAGE and MALDI-TOF mass spectrometry were used to identify the compounds in this venom responsible for the cytotoxic effects. In general, viper venoms were potently cytotoxic, with MCF-7 cells showing greater sensitivity, while elapid and colubrid venoms were much less toxic; notable exceptions included the elapid genera Micrurus, Naja and Pseudechis, which were quite cytotoxic to both cell lines. However, venoms with the most potent cytotoxicity were often not those with low mouse LD50s, including some dangerously venomous viperids and Australian elapids. This study confirmed that many venoms contain cytotoxic compounds, including catalytic PLA2s, and several venoms also showed significant differential toxicity toward the two cancer cell lines. Our results indicate that several previously uncharacterized venoms could contain promising lead compounds for drug development.


Colubridae Cytotoxicity Drug development Melanoma Phospholipase A2 Three-finger toxin 



Funding for this project was provided by a grant (to SPM) from the Colorado Office of Economic Development and Trade (COEDIT), Bioscience Discovery Evaluation Grant Program. Additional funding was provided by the UNC Office of Sponsored Programs. We thank Peter J. Mirtschin of Venom Supplies Pty. Ltd. for providing P. porphyriacus venom, Dr. Charlotte Ownby of Oklahoma State University for numerous elapid and viperid venoms, and A. Ah-Young, B. Heyborne, J. LeRoy Waite, and A. Wastell for assistance with venom extractions (rattlesnakes and colubrids).

Conflict of interest

The authors state that there are no conflicts of interest.

Ethics Standard

All vertebrate animal manipulations (venom extractions of snakes) were in accordance with protocols approved by the UNC IACUC.


  1. Alape-Girón A, Sanz L, Escolano J, Flores-Díaz M, Madrigal M, Sasa M, Calvete JJ (2008) Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations. J Proteome Res 7:3556–3571CrossRefGoogle Scholar
  2. Anderson LA, Dufton MJ (1998) Acetylcholinesterases. In: Bailey GS (ed) Enzymes from snake venoms. Alaken, Ft. Collins, pp 545–578Google Scholar
  3. Barlow A, Pook CE, Harrison RA, Wüster W (2009) Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proc Biol Sci 276:2443–2449CrossRefGoogle Scholar
  4. Brown MC, Staniszewska I, Valle LD, Tuszynski GP, Marcinkiewicz C (2008) Angiostatic activity of obtustatin as alpha1beta1 integrin inhibitor in experimental melanoma growth. Int J Cancer 123:2195–2203CrossRefGoogle Scholar
  5. Calvete JJ (2010) Snake venomics, antivenomics, and venom phenotyping: the ménage à trois of proteomic tools aimed at understanding the biodiversity of venoms. In: Kini RM, Clemetson KJ, Markland FS, McLane MA, Morita T (eds) Toxins and hemostasis: from bench to bedside. Springer, Dordrecht, pp 45–72CrossRefGoogle Scholar
  6. Calvete JJ, Marcinkiewicz C, Monleon D, Esteve V, Celda B, Juarez P, Sanz L (2005) Snake venom disintegrins: evolution of structure and function. Toxicon 45:1063–1074CrossRefGoogle Scholar
  7. Calvete JJ, Marcinkiewicz C, Sanz L (2006) Snake venomics of Bitis gabonica gabonica. Protein family composition, subunit organization of venom toxins, and characterization of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2. J Proteome Res 6:326–336CrossRefGoogle Scholar
  8. Chaim-Matyas A, Ovadia M (1987) Cytotoxic activity of various snake venoms on melanoma, B16F10 and chondrosarcoma. Life Sci 40:1601–1607CrossRefGoogle Scholar
  9. Doley R, Pahari S, Mackessy SP, Kini RM (2008) Accelerated exchange of exon segments in viperid three-finger toxin genes (Sistrurus catenatus edwardsii; Desert Massasauga). BMC Evol Biol 8:196CrossRefGoogle Scholar
  10. Doley R, Zhou X, Kini RM (2010) Snake venom phospholipase A2 enzymes. In: Mackessy SP (ed) Handbook of venoms and toxins of reptiles. Taylor and Francis/CRC Press, Boca Raton, pp 173–206Google Scholar
  11. Earl STH, Masci PP, Jersey JD, Lavin MF, Dixon J (2012) Drug development from Australian elapid snake venoms and the Venomics pipeline of candidates for haemostasis: Textilinin-1 (Q8008), Haempatch™ (Q8009) and CoVase™ (V0801). Toxicon 59:456–463CrossRefGoogle Scholar
  12. El-Refael MF, Sarkar NH (2009) Snake venom inhibits the growth of mouse mammary tumor cells in vitro and in vivo. Toxicon 54:33–41CrossRefGoogle Scholar
  13. Ferreira SH (1965) A bradykinin-potentiating factor (BPF) present in the venom of Bothrops jararaca. Br J Pharmacol Chemother 24:163–169CrossRefGoogle Scholar
  14. Ferreira SH, Bartelt DC, Greene LJ (1970) Isolation of bradykinin-potentiating peptides from Bothrops jararaca venom. Biochemistry 9:2583–2593CrossRefGoogle Scholar
  15. Fox JW, Serrano SM (2005) Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases. Toxicon 45:969–985CrossRefGoogle Scholar
  16. Fox JW, Serrano SM (2007) Approaching the golden age of natural product pharmaceuticals from venom libraries: an overview of toxins and toxin-derivatives currently involved in therapeutic or diagnostic applications. Curr Pharm Des 13:2927–2934CrossRefGoogle Scholar
  17. Fox JW, Serrano SMT (2010) Snake venom metalloproteinases. In: Mackessy SP (ed) Handbook of venoms and toxins of reptiles. Taylor and Francis/CRC Press, Boca Raton, pp 115–138Google Scholar
  18. Fruchart-Gaillard C, Mourier G, Blanchet G, Vera L, Gilles N, Ménez R, Marcon E, Stura EA, Servent D (2012) Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors. PLoS One 7:39166CrossRefGoogle Scholar
  19. Gauthier JA, Kearney M, Maisano JA, Rieppel O, Behlke ADB (2012) Assembling the squamate tree of life: perspectives from the phenotype and the fossil record. Bull Peabody Mus Nat Hist 53:3–308CrossRefGoogle Scholar
  20. Gibbs HL, Mackessy SP (2009) Functional basis of a molecular adaptation: prey-specific toxic effects of venom from Sistrurus rattlesnakes. Toxicon 53:672–679CrossRefGoogle Scholar
  21. Gutiérrez JM, Lomonte B, León G, Alape-Girón A, Flores-Díaz M, Sanz L, Angulo Y, Calvete JJ (2009) Snake venomics and antivenomics: proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming. J Proteomics 72:165–182CrossRefGoogle Scholar
  22. Heyborne WH, Mackessy SP (2013) Isolation and characterization of a taxon-specific three-finger toxin from the venom of the Green Vinesnake (Oxybelis fulgidus; family Colubridae). Biochimie 95:1923–1932CrossRefGoogle Scholar
  23. Hill RE, Mackessy SP (1997) Venom yields from several species of colubrid snakes and differential effects of ketamine. Toxicon 35:671–678CrossRefGoogle Scholar
  24. Huang P, Mackessy SP (2004) Biochemical characterization of phospholipase A2 (trimorphin) from the venom of the Sonoran Lyre Snake Trimorphodon biscutatus lambda (family Colubridae). Toxicon 44:27–36CrossRefGoogle Scholar
  25. Huguet EL, McMahon JA, McMahon AP, Bicknell R, Harris AL (1994) Differential expression of human Wnt genes 2, 3, 4, and 7B in human breast cell lines and normal and disease states of human breast tissue. Cancer Res 54:2615–2621Google Scholar
  26. Kamiguti A, Zuzel M, Theakston R (1998) Snake venom metalloproteinases and disintegrins: interactions with cells. Braz J Med Biol Res 31:853–862CrossRefGoogle Scholar
  27. Kereiakes DJ, Kleiman NS, Ambrose J, Cohen M, Rodriguez S, Palabrica T, Herrmann TC, Sutton JM, Weaver WD, McKee DB, Fitzpatrick V, Sax FL, Higby N, Ratner D, Slatylak S, DeAngelo, D, Trainor K, Rose D, Johnson S, Miele R, Cowfer J, Martin J (1996) Randomized, double-blind, placebo-controlled dose-ranging study of tirofiban (MK-383) platelet IIb/IIIa blockade in high risk patients undergoing coronary angioplasty. J Am Coll Cardiol 27:536–542Google Scholar
  28. Kini RM (2002) Molecular molds with multiple missions: functional sites in three-finger toxins. Clin Exp Pharmacol Physiol 29:815–822CrossRefGoogle Scholar
  29. Kini RM (2003) Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon 42:827–840CrossRefGoogle Scholar
  30. Kini RM, Doley R (2010) Structure, function and evolution of three-finger toxins: mini proteins with multiple targets. Toxicon 56(2010):855–867CrossRefGoogle Scholar
  31. Koh CY, Kini RM (2012) From snake venom toxins to therapeutics–cardiovascular examples. Toxicon 59:497–506CrossRefGoogle Scholar
  32. Lin E, Wang Q, Swenson S, Jadvar H, Groshen S, Ye W, Markland FS, Pinski J (2010) The disintegrin contortrostatin in combination with docetaxel is a potent inhibitor of prostate cancer in vitro and in vivo. Prostate 70:1359–1370Google Scholar
  33. Lomonte B, Tsai WC, Ureña-Diaz JM, Sanz L, Mora-Obando D, Sánchez EE, Fry BG, Gutiérrez JM, Gibbs HL, Sovic MG, Calvete JJ (2014) Venomics of New World pit vipers: genus-wide comparisons of venom proteomes across Agkistrodon. J Proteomics 96:103–116CrossRefGoogle Scholar
  34. Lucena S, Sanchez EE, Perez JC (2011) Anti-metastatic activity of the recombinant disintegrin, r-mojastin 1, from the Mohave rattlesnake. Toxicon 57:794–802CrossRefGoogle Scholar
  35. Mackessy SP (1988) Venom ontogeny in the Pacific rattlesnakes Crotalus viridis helleri and C. v. oreganus. Copeia 1988:92–101CrossRefGoogle Scholar
  36. Mackessy SP (1993) Fibrinogenolytic proteases from the venoms of juvenile and adult northern Pacific rattlesnake (Crotalus viridis oreganus). Comp Biochem Physiol 106B:181–189Google Scholar
  37. Mackessy SP (1998) Phosphodiesterases, ribonucleases and deoxyribonucleases. In: Bailey GS (ed) Enzymes from snake venoms. Alaken, Ft. Collins, pp 361–404Google Scholar
  38. Mackessy SP (2002) Biochemistry and pharmacology of colubrid snake venoms. J Toxicol Toxin Rev 21:43–83CrossRefGoogle Scholar
  39. Mackessy SP (2008) Venom composition in rattlesnakes: trends and biological significance. In: Hayes WK, Cardwell MD, Beaman KR, Bush SP (eds) The biology of rattlesnakes. Loma Linda University Press, Loma Linda, pp 495–510Google Scholar
  40. Mackessy SP (2010a) The field of reptile toxinology. Snakes, lizards, and their venoms. In: Mackessy SP (ed) Handbook of venoms and toxins of reptiles. Taylor and Francis/CRC Press, Boca Raton, pp 3–24Google Scholar
  41. Mackessy SP (2010b) The evolution of venom composition in the Western Rattlesnakes (Crotalus viridis sensu lato): toxicity versus tenderizers. Toxicon 55:1463–1474CrossRefGoogle Scholar
  42. Mackessy SP, Williams K, Ashton K (2003) Characterization of the venom of the midget faded rattlesnake (Crotalus viridis concolor): a case of venom paedomorphosis? Copeia 2003:769–782CrossRefGoogle Scholar
  43. Mackessy SP, Sixberry NM, Heyborne WH, Fritts T (2006) Venom of the Brown Treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 47:537–548CrossRefGoogle Scholar
  44. Masuda S, Hayashi H, Araki S (1998) Two vascular apoptosis-inducing proteins from snake venom are members of the metalloprotease/disintegrin family. Eur J Biochem 253:36–41CrossRefGoogle Scholar
  45. Masuda S, Ohta T, Kaji K, Fox JW, Hayashi H, Araki S (2000) cDNA cloning and characterization of vascular apoptosis-inducing protein 1. Biochem Biophys Res Commun 278:197–204CrossRefGoogle Scholar
  46. Masuda S, Hayashi H, Atoda H, Morita T, Araki S (2001) Purification, cDNA cloning and characterization of the vascular apoptosis-inducing protein, HV1, from Trimeresurus flavoviridis. Eur J Biochem 268:3339–3345CrossRefGoogle Scholar
  47. McLane MA, Joerger T, Mahmoud A (2008) Disintegrins in health and disease. Front Biosci 13:6617–6637CrossRefGoogle Scholar
  48. Minton SA, Weinstein SA (1986) Geographic and ontogenic variation in venom of the western diamondback rattlesnake Crotalus atrox. Toxicon 24:71–80CrossRefGoogle Scholar
  49. Mirtschin PJ, Crowe GR, Davis R (1990) Dangerous snakes of Australia. In: Gopalakrishnakone P, Chou LM (eds) Snakes of medical importance (Asia-Pacific Region). Venom and Toxin Research Group, NUS, Singapore, pp 1–174Google Scholar
  50. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  51. Mukherjee AK (2014) A major phospholipase A2 from Daboia russelii russelii venom shows potent anticoagulant action via thrombin inhibition and binding with plasma phospholipids. Biochimie 99:153–161CrossRefGoogle Scholar
  52. Mukherjee AK, Mackessy SP (2013) Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell’s Viper Daboia russelii russelii and assessment of its therapeutic potential. BBA Gen Subj 1830:3476–3488CrossRefGoogle Scholar
  53. Nirthanan S, Gwee MCE (2004) Three-finger neurotoxins and the nicotinic acetylcholine receptor, forty years on. J Pharmacol Sci 94:1–17CrossRefGoogle Scholar
  54. Núñez V, Cid P, Sanz L, De La Torre P, Angulo Y, Lomonte B, Gutiérrez JM, Calvete JJ (2009) Snake venomics and antivenomics of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil, Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism. J Proteomics 73:57–78CrossRefGoogle Scholar
  55. Öhler M, Georgieva D, Seifert J, von Bergen M, Arni RK, Genov N, Betzel C (2010) The venomics of Bothrops alternatus is a pool of acidic proteins with predominant hemorrhagic and coagulopathic activities. J Proteome Res 9:2422–2437CrossRefGoogle Scholar
  56. Oron U, Chaim-Matyas A, Ovadia M (1992) Histopathological changes in WEHI-3B leukemia cells following intoxication by cytotoxin P4 from Naja nigricollis nigricollis venom. Toxicon 30:1122–1126CrossRefGoogle Scholar
  57. Pal SK, Gomes A, Dasgupta SC, Gomes A (2002) Snake venom as therapeutic agents: from toxin to drug development. Indian J Exp Biol 40:1353–1358Google Scholar
  58. Pawlak J, Mackessy SP, Fry BG, Bhatia M, Mourier G, Fruchart-Gaillard C, Servent D, Ménez R, Stura E, Ménez A, Kini RM (2006) Denmotoxin: a three-finger toxin from colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity. J Biol Chem 281:29030–29041CrossRefGoogle Scholar
  59. Pawlak J, Mackessy SP, Sixberry NM, Stura EA, Le Du MH, Ménez R, Foo CS, Ménez A, Nirthanan S, Kini RM (2009) Irditoxin, a novel covalently linked heterodimeric three-finger toxin with high taxon-specific neurotoxicity. FASEB J 23:534–545CrossRefGoogle Scholar
  60. Pyron RA, Burbrink FT, Wiens JJ (2013) A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evol Biol 13:93CrossRefGoogle Scholar
  61. Samel M, Trummal K, Siigur E, Siigur J (2012) Effect of HUVEC apoptosis inducing proteinase from Vipera lebetina venom (VLAIP) on viability of cancer cells and on platelet aggregation. Toxicon 60:648–655CrossRefGoogle Scholar
  62. Sánchez EE, Rodríguez-Acosta A, Palomar R, Lucena SE, Bashir S, Soto JG, Pérez JC (2009) Colombistatin: a disintegrin isolated from the venom of the South American snake (Bothrops colombiensis) that effectively inhibits platelet aggregation and SK-Mel-28 cell adhesion. Arch Toxicol 83:271–279CrossRefGoogle Scholar
  63. Saviola AJ, Chiszar D, Busch C, Mackessy SP (2013) Molecular basis for prey relocation in viperid snakes. BMC Biol 11:20CrossRefGoogle Scholar
  64. Saviola AJ, Peichoto ME, Mackessy SP (2014) Rear-fanged snake venoms: an untapped source of novel compounds and potential drug leads. Toxin Rev. doi:  10.3109/15569543.2014.942040. (in press)
  65. Sharma SD, Jiang J, Hadley ME, Bentley DL, Hruby VJ (1996) Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropinreceptors. Proc Natl Acad Sci 93:13715–13720CrossRefGoogle Scholar
  66. St. Pierre L, Fischer H, Adams DJ, Schenning M, Lavidis N, de Jersey J, Masci PP, Lavin MF (2007) Distinct activities of novel neurotoxins from Australian venomous snakes for nicotinic acetylcholine receptors. Cell Mol Life Sci 64:2829–2840CrossRefGoogle Scholar
  67. Swenson S, Costa F, Ernst W, Fujii G, Markland F (2005) Contortrastatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity. Pathophysiol Haemost Thromb 34:169–176CrossRefGoogle Scholar
  68. Takacs Z, Nathan S (2014) Animal venoms in medicine. In: Wexler P (ed) Encyclopedia of toxicology, Third edn. Elsevier, Amsterdam, pp 252–529. doi: 10.1016/B978-0-12-386454-3.01241-0 CrossRefGoogle Scholar
  69. Takahashi K, Suzuki K (1993) Association of insulin-like growth-factor-I-induced DNA synthesis with phosphorylation and nuclear exclusion of p53 in human breast cancer MCF-7 cells. Int J Cancer 55:453–458CrossRefGoogle Scholar
  70. Tian J, Paquette-Straub C, Sage EH, Funk SE, Patel V, Galileo D, McLane MA (2007) Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin. Toxicon 49:899–908CrossRefGoogle Scholar
  71. Vink S, Jin AH, Poth KJ, Head GA, Alewood PF (2012) Natriuretic peptide drug leads from snake venom. Toxicon 59:434–445CrossRefGoogle Scholar
  72. Vonk FJ, Jackson K, Doley R, Madaras F, Mirtschin PJ, Vidal N (2011) Snake venom: from fieldwork to the clinic. BioEssays 33:269–279CrossRefGoogle Scholar
  73. Wiens JJ, Hutter CR, Mulcahy DG, Noonan BP, Townsend TM, Sites JW Jr, Reeder TW (2012) Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species. Biol Lett 8:1043–1046CrossRefGoogle Scholar
  74. Yalcın HT, Ozen MO, Gocmen B, Nalbantsoy A (2014) Effect of Ottoman viper (Montivipera xanthina (Gray, 1849)) venom on various cancer cells and on microorganisms. Cytotechnology 66:87–94CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Michael J. Bradshaw
    • 1
  • Anthony J. Saviola
    • 1
  • Elizabeth Fesler
    • 1
  • Stephen P. Mackessy
    • 1
    Email author
  1. 1.School of Biological SciencesUniversity of Northern ColoradoGreeleyUSA

Personalised recommendations